When this research started, there were no known steroid phosphoesters. The first paper used Roman numerals to distinguish the four new compounds that had been discovered. We have now identified more than 20 different steroid esters and each one can exist as either a phosphoethanolamine or a phosphocholine ester. In addition, on mass spectroscopy each phosphocholine ester can be present in one of four forms, designated A, B, C, or D as shown in the table.
Serum samples have low levels of D because serum K+ levels are low. S fragments can be hard to detect in serum without additional applied voltage. S fragments are readily detected after desalting and for phosphoethanolamine esters.
The phosphocholine fragment occurs at m/z=184 Da. It is readily detected in purified compounds, but it is not useful because of the presence of phosphatidyl lipids in serum and tissues, most of which generate a fragment ion at m/z=184 Da.
With this background, it is possible to examine a mass spectrogram and identify the phosphocholine esters, including the m/z of the S fragment.
The proposed symbols for each phosphoester steroid compound are Cxxx where C indicates it is a phosphocholine ester and xxx is the m/z of the S fragment. Exxx would indicate it is a phosphoethanolamine ester. Exxx compound will have m/z =42 Da lower than the corresponding Cxxx compound with a similar S fragment. The intact molecular mass of S is usually 17 Da higher than the observed fragment. For carboxylic acid intermediates, S is only 15 Da higher than the observed S fragment. The extra hydrogen atoms were needed to generate a cation. An asterisk added to the symbol (*Cxxx) indicates that the steroid seems to be a carboxylic acid.
Investigators with access to a mass spectrometer can identify each compound without reference to specificity of antibodies generated to digoxin, ouabain or other cardiotonic glycosides.